Modulators of P-selectin glycoprotein ligand 1

ABSTRACT

Multimeric compounds that bind to P-Selectin Glycoprotein 1 (PSGL-1) on the surface of T cells or natural killer (NK) cells can be used to induce T cell or NK cell depletion and/or to induce T cell or NK cell apoptosis. The multimeric compounds and methods of the invention can be used to control unwanted T cell- or NK cell-mediated immune responses in conditions such as inflammatory diseases, autoimmune diseases, transplant rejection, and allergic diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 10/051,497, filed Jan. 18, 2002, which claims priority of U.S.Application No. 60/310,196, filed Aug. 3, 2001. The prior applicationsare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] The invention relates to compositions and methods for controllingimmune responses.

BACKGROUND OF THE INVENTION

[0003] The control of unwanted immune responses is a critical issue inthe treatment of diseases such as inflammatory diseases, autoimmunediseases, transplant rejection, allergic diseases and T cell-derivedcancers. The activity of overly aggressive T cells can be controlled byimmunosuppression or by the induction of immunological tolerance.Tolerance is defined as a state where the immune system is madeunresponsive to an antigen, whereas immunosuppression, which decreasesthe immune response to antigens, usually requires the continued use ofmedication. In organ transplantation, T cells play an essential role inthe immune response to alloantigens. Current immunosuppressive regimescommonly involve the use of corticosteroid, cyclosporin or rapamycin,which block the transcription of IL-2, a key growth factor for T cells,or inhibit IL-2 dependent proliferation. However, a number of monoclonalantibodies which either act as T cell-depleting agents (e.g. CD3, CD4,CD8), or as inhibitors of the cytokine signaling or the co-stimulatorypathways of T cells (e.g. CD25, B7-1, B7-2, CD152, CTLA4) havedemonstrated effectiveness in reducing the incidence of rejection withlimited side effects or toxicity. Some of these agents have been shownto have some degree of success in treating autoimmune disease and inprolonging graft survival.

[0004] Apoptosis is widely believed to be of vital importance formaintaining the proper function of the immune system and a majormechanism to remove unwanted cells (Kabelitz et al. Immunol. Today14:338-340 (1993); Raff, Nature:356:397-399 (1992)). Various signalsoriginating from either inside or outside a cell influence the life anddeath of the cell. Antibodies against T cell surface molecules such asFas (or CD95, MW=43 kD), TNFR2 (MW=75 kD), CD2 (MW=45 kD) and CTLA-4(MW=33 kD) induce the apoptosis of T cells (Osborne, Curr. Opin.Immunol. 8:245-248 (1996); Lin et al. J. Immunol. 158:598-603 (1997);Zhang et al. Nature:377:348-350 (1995); Lai et al. Eur. J. Immunol.25:3243-3248 (1995); Mollereau et al. J. Immunol. 156:3184-3190 (1996);Gribben et al. Proc. Natl. Acad. Sci. USA 92:811-815 (1995)). Attemptsto use Fas and TNFR2 molecules to control unwanted T cells have beenhampered by the fact that these two molecules are expressed not only onimmune cells, but also on several other important organ systems likeliver. This expression pattern potentially limits the therapeuticapplication of these two antibodies (Ogasawara et al. Nature 364:806-809(1993); Pfeffer et al. Cell:73:457-467 (1993); Engelmann et al. J.Biological Chemistry 265:14497-14504 (1990)).

[0005] Selectins, integrins and immunoglobulin (Ig) superfamily membersare three major classes of adhesion molecules that are important to theinteraction of leukocytes and platelets either with themselves or withthe extracellular matrix and vascular endothelium (Springer, Nature346:425 (1990); Osborn, Cell 62:3 (1990); Hynes, Cell 69:11 (1992)). Anadhesion molecule on one cell type often binds to another adhesionmolecule expressed on a different cell type, forming a ligand-receptorpair.

[0006] The selectin family consists of P-selectin (also known as CD62,CD62P, GMP140, and PADGEM), E-selectin (also known as ELAM-1 and CD62E),and L-selectin (also known as LECAM-1, Mel-14, LAM-1, and CD62L). Theselectins are highly homologous, composed of a 120 amino acid N-terminallectin domain, an EGF-like domain, a variable number of multiple shortconsensus repeat (SCR) domains homologous to those found in complementregulatory proteins, followed by a transmembrane domain and shortcytoplasmic tail (Siegelman et al., Science 243:1165-1172 (1989); Laskyet al., Cell 56:1045-1055 (1989); Tedder et al., J. Exp. Med.170:123-133 (1989); Johnson et al., Cell 56:1033-1044 (1989); Bevilacquaet al., Proc. Natl. Acad. Sci. USA 84:9238-9242 (1987), Bevilacqua etal., Science 243:1160-1165 (1989), Bevilacqua et al., J. Clin. Invest.91:379-387 (1993), Camerini et al., Nature 280:496-498 (1989)). Theselectins have overlapping but distinct specificities for cell surfacereceptors (Bevilacqua et al., J. Clin. Invest. 91:379-387 (1993); Feize,Current Opinion in Struct. Biol. 3:701-710 (1993); Berg et al., Biochem.Biophys. Res. Comm. 184:1048-1055 (1992); Foxall et al., J. Cell Biol.117:895-902 (1992); Larsen et al., J. Biol. Chem. 267:11104-11110(1992); Polley et al., Proc. Natl. Acad. Sci. USA 88:6224-6228 (1991)).

[0007] P-selectin, E-selectin, and L-selectin mediate the firstleukocyte-endothelial cell and platelet-leukocyte adhesive interactionsduring inflammation (Bevilacqua et al., 1993, supra). All threeselectins have been demonstrated to participate in an initial “rolling”interaction of leukocytes with activated endothelium (von Andrian etal., Proc. Natl. Acad. Sci. USA 88:7538-7542 (1991); Ley et al., Blood77:2553-2555 (1991); Abassi et al., J. Clin. Invest. 92:2719-2730(1993); Dore et al., Blood 82:1308-1316 (1993); Jones et al., Biophys.J. 65:1560-1569 (1993); Mayadas et al., Cell 74:541-554 (1993)).P-selectin, expressed on activated platelets and endothelial cells,binds to cell surface proteins on most leukocytes (McEver et al., J.Biol. Chem. 250:9799-9804 (1984); Hsu-Lin et al., J. Biol. Chem.264:8121-9126 (1984)). E-selectin, expressed on cytokine-activatedendothelial cells (e.g., after TNF-alpha or IL-1 stimulation for 6-8hours) binds to cell surface proteins on most leukocytes (McEver et al.,J. Clin. Invest. 100:485-492 (1997); Bevilacqua et al., 1987, supra;Bevilacqua et al., 1989, supra). L-selectin, expressed on mostleukocytes, binds to cell surface proteins on some endothelial cells andon other leukocytes (Gallatin et al., Nature 304:30-34 (1983); Berg etal., Immunol. Rev. 108:5-18 (1989); Berg et al., J. Cell. Biol.114:343-349 (1991), Hallman et al., Biochem. Biophys. Res. Comm.174:236-243 (1991); Smith et al., J. Clin. Invest. 87:609-618 (1991);Spertini et al., J. Immunol. 147:2565-2573 (1991)). All three selectinshave been shown to bind to a cell surface protein, PSGL-1, whoseexpression is largely limited to leukocytes, and in particular T cellsand NK cells. Posttranslational modifications of PSGL-1 are required forbinding to P-selectin, E-selectin, and L-selectin (McEver et al., J.Clin. Invest., 1997, supra).

SUMMARY OF THE INVENTION

[0008] The invention is based on the discovery that T cells can bedepleted and/or induced to undergo apoptosis by the engagement of the Tcell surface antigen P-Selectin Glycoprotein Ligand-1 (PSGL-1). T celldepletion can be particularly useful for the treatment of conditionsassociated with an excessive or unwanted T cell-mediated immune responseor excessive or unwanted T cell proliferation. For example, thedepletion of T cells can cause the reduction or elimination ofundesirable T cell activity or proliferation associated withinflammatory diseases, autoimmune diseases, transplant rejection,allergic diseases, and/or T cell-derived cancers. The inventionencompasses methods of using modulators of PSGL-1 function to prevent orreduce a T cell-mediated immune response as well as methods of screeningfor modulators of PSGL-1 function.

[0009] In one aspect, the invention features a method of preventing orreducing a T cell-mediated immune response in an individual. The methodincludes the following steps: selecting an individual diagnosed ashaving or as being at risk of acquiring a condition characterized by anexcessive or unwanted T cell-mediated immune response; and administeringto the individual a compound that binds to PSGL-1 on the surface of a Tcell, wherein the binding of the compound to PSGL-1 on the surface ofthe T cell induces a signal transduction pathway that results in thedeath of the T cell, thereby preventing or reducing a T cell-mediatedimmune response in the individual.

[0010] The compound used in such a method can include an antibody orantigen binding fragment thereof that specifically binds to PSGL-1. Inone example, the compound is a monoclonal antibody that specificallybinds to PSGL-1. In one embodiment, the method includes an additionalstep of administering an agent that binds to the monoclonal antibody andinduces the cross-linking of a plurality of PSGL-1 antigens on thesurface of the T cell.

[0011] In some embodiments, the method includes inducing thecross-linking of a plurality of PSGL-1 antigens on the surface of the Tcell, wherein the cross-linking induces the signal transduction pathwaythat results in the death of the T cell.

[0012] In some embodiments, the method includes the following steps: (i)selecting an individual diagnosed as having or as being at risk ofacquiring a condition characterized by an excessive or unwanted Tcell-mediated immune response; and (ii) administering to the individuala multimeric compound that binds to at least two PSGL-1 proteins on thesurface of a T cell, wherein the multimeric compound contains twopolypeptide chains, each of the polypeptide chains including (a) abinding domain that binds to PSGL-1, and (b) a heterologous amino acidsequence, wherein the polypeptide chains are linked via the heterologousamino acid sequence to form the multimeric compound, and wherein thebinding of the multimeric compound to the at least two PSGL-1 proteinson the surface of the T cell induces a signal transduction pathway thatresults in the death of the T cell, thereby preventing or reducing a Tcell-mediated immune response in the individual.

[0013] The multimeric compound can be a homo-multimeric compound or ahetero-multimeric compound. The binding domain can optionally contain aP-Selectin extracellular domain or a PSGL-1-binding fragment thereof, anE-Selectin extracellular domain or a PSGL-1-binding fragment thereof, anL-Selectin extracellular domain or a PSGL-1-binding fragment thereof, ananti-PSGL-1 antibody or a PSGL-1-binding fragment thereof, a PSGL-1binding polypeptide selected from a phage display library, or acombination of any of the above.

[0014] In certain embodiments, the multimeric compound does not includean anti-PSGL-1 antibody or an antibody fragment that binds to PSGL-1.

[0015] The heterologous amino acid sequence can optionally contain acell surface receptor binding region, e.g., an immunoglobulin heavychain constant region. In some embodiments, the polypeptide chains arecovalently linked, e.g., disulfide linked, via the heterologous aminoacid sequence to form the multimeric compound.

[0016] In certain embodiments, the method can include an additional stepof administering to the individual an agent that binds to the multimericcompound via the heterologous amino acid sequence and inducescross-linking of a plurality of PSGL-1 antigens on the surface of the Tcell.

[0017] In some embodiments, a method described herein includes a step ofselecting an individual diagnosed as having an autoimmune disease. Inanother example, the method includes a step of selecting an individualdiagnosed as having an inflammatory disease. In another example, themethod includes a step of selecting an individual that has received oris expected to receive an allogeneic or xenogeneic transplant. Inanother example, the method includes a step of selecting an individualdiagnosed as having an allergic disease. In another example, the methodincludes a step of selecting an individual diagnosed as having a T cellcancer.

[0018] In some embodiments, the T cell is an activated T cell. In oneexample, the T cell is a CD4+ T cell. In another example, the T cell isa CD8+ T cell.

[0019] In some embodiments, the method includes a step of detecting thenumber of T cells in a first biological sample taken from the individualbefore the administration of the compound (e.g., a multimeric compound)and comparing the results with the number of T cells in a secondbiological sample taken from the individual after the administration ofthe compound (e.g., a multimeric compound).

[0020] In some embodiments, the method includes a step of detecting abiological activity of T cells in a first biological sample taken fromthe individual before the administration of the compound (e.g., amultimeric compound) and comparing the results with the biologicalactivity of T cells in a second biological sample taken from theindividual after the administration of the compound (e.g., a multimericcompound).

[0021] In some embodiments, the administration results in the depletionof at least 10% of activated T cells in the individual. In someembodiments, the administration results in the depletion of at least10%, 20%, 30%, 40%, 50%, or more of the activated T cells in theindividual.

[0022] In some embodiments, the antibody or antigen binding fragmentthereof or the multimeric compound induces the death of at least 10% ofactivated T cells in the individual after exposure to the antibody orantigen binding fragment thereof or the multimeric compound. In someembodiments, the administration induces the death of at least 10%, 20%,30%, 40%, 50%, or more of the activated T cells in the individual. Celldeath can be measured at any time, e.g., one, two, three, four, five,six, seven, or more days after exposure to the antibody or antigenbinding fragment thereof or the multimeric compound.

[0023] In another aspect, the invention features a method of inducingthe death of a T cell or a natural killer (NK) cell. The method includesthe steps of: providing a T cell or NK cell expressing PSGL-1 on itscell surface; and contacting the T cell or NK cell with a compound thatbinds to PSGL-1 on the surface of the T cell or NK cell, wherein thebinding of the compound to PSGL-1 on the surface of the T cell or NKcell induces a signal transduction pathway that results in the death ofthe T cell or NK cell.

[0024] The compound used in such a method can include an antibody orantigen binding fragment thereof that specifically binds to PSGL-1. Inone example, the compound is a monoclonal antibody that specificallybinds to PSGL-1. In one embodiment, the method includes a step ofcontacting the monoclonal antibody with an agent that binds to themonoclonal antibody and induces the cross-linking of a plurality ofPSGL-1 antigens on the surface of the T cell or NK cell.

[0025] In one embodiment, the method includes the following steps: (i)providing a T cell or NK cell expressing PSGL-1 on its cell surface; and(ii) contacting the T cell or NK cell with a multimeric compound thatbinds to at least two PSGL-1 proteins on the surface of the T cell or NKcell, wherein the multimeric compound contains two polypeptide chains,each of the polypeptide chains including (a) a binding domain that bindsto PSGL-1, and (b) a heterologous amino acid sequence, wherein thepolypeptide chains are linked via the heterologous amino acid sequenceto form the multimeric compound, wherein the binding of the multimericcompound to the at least two PSGL-1 proteins on the surface of the Tcell or NK cell induces a signal transduction pathway that results inthe death of the T cell or NK cell.

[0026] The multimeric compound can be a homo-multimeric compound or ahetero-multimeric compound. The binding domain can optionally contain aP-Selectin extracellular domain or a PSGL-1-binding fragment thereof, anE-Selectin extracellular domain or a PSGL-1-binding fragment thereof, anL-Selectin extracellular domain or a PSGL-1-binding fragment thereof, ananti-PSGL-1 antibody or a PSGL-1-binding fragment thereof, a peptideselected from a phage display library, or a combination of any of theabove.

[0027] The heterologous amino acid sequence can optionally contain acell surface receptor binding region, e.g., an immunoglobulin heavychain constant region. In some embodiments, the polypeptide chains arecovalently linked, e.g., disulfide linked, via the heterologous aminoacid sequence to form the multimeric compound.

[0028] In some embodiments, the method includes an additional step ofcontacting the multimeric compound with an agent that binds to themultimeric compound via the heterologous amino acid sequence and inducescross-linking of a plurality of PSGL-1 antigens on the surface of the Tcell.

[0029] In some embodiments, the method includes a step of inducing thecross-linking of a plurality of PSGL-1 antigens on the surface of the Tcell or NK cell, wherein the cross-linking induces the signaltransduction pathway that results in the death of the T cell or NK cell.

[0030] In some embodiments of methods described herein, the T cell is anactivated T cell. In one example, the T cell is a CD4+ T cell. Inanother example, the T cell is a CD8+ T cell.

[0031] In some embodiments of methods described herein, the methodincludes a step of assessing the viability of the T cell or NK cellafter the contacting with the compound (e.g., a multimeric compound).

[0032] In some embodiments of methods described herein, the methodincludes a step of assessing a biological activity of the T cell or NKcell after the contacting with the compound (e.g., a multimericcompound).

[0033] In some embodiments, the method includes inducing the death of anactivated T cell.

[0034] In another aspect, the invention features a method of screeningfor a modulator of PSGL-1 function. The method includes the steps of:providing a cell expressing PSGL-1 on the surface of the cell;contacting the cell with a test substance; and measuring the viabilityof the cell after contacting the cell with the test substance to therebydetermine if the test substance is a modulator of PSGL-1 function.

[0035] In one embodiment, the method includes the step of detecting thedeath of the cell induced by the test substance to thereby determinethat the test substance is a modulator of PSGL-1 function.

[0036] In one embodiment, the test substance is an antibody or antigenbinding fragment thereof that specifically binds to PSGL-1. In oneexample, the test substance is a monoclonal antibody that specificallybinds to PSGL-1. In one embodiment, the method includes the step ofcontacting the monoclonal antibody with an agent that binds to themonoclonal antibody and induces the cross-linking of a plurality ofPSGL-1 antigens on the surface of the cell.

[0037] In one embodiment, the method includes the step of inducing thecross-linking of a plurality of PSGL-1 antigens on the surface of thecell, wherein the cross-linking induces the signal transduction pathwaythat results in the death of the cell.

[0038] In one embodiment, the T cell is an activated T cell. In oneexample, the T cell is a CD4+ T cell. In another example, the T cell isa CD8+ T cell.

[0039] In one embodiment, the method includes the step of manufacturingbulk quantities of the test substance and formulating the test substancein a pharmaceutically acceptable carrier.

[0040] In another aspect, the invention features a kit containing: acompound that binds to PSGL-1 on the surface of a T cell, wherein thebinding of the compound to PSGL-1 on the surface of the T cell induces asignal transduction pathway that results in the death of the T cell; andinstructions for use of the compound to treat a condition associatedwith an excessive or unwanted T cell mediated immune response orexcessive or unwanted T cell proliferation such as inflammation,autoimmunity, transplant rejection, an allergic condition, or a T cellcancer.

[0041] In one embodiment, the kit contains: (i) a multimeric compoundthat binds to at least two PSGL-1 proteins on the surface of a T cell,wherein the multimeric compound contains two polypeptide chains, each ofthe polypeptide chains including (a) a binding domain that binds toPSGL-1, and (b) a heterologous amino acid sequence, wherein thepolypeptide chains are linked via the heterologous amino acid sequenceto form the multimeric compound, wherein the binding of the multimericcompound to the at least two PSGL-1 proteins on the surface of the Tcell induces a signal transduction pathway that results in the death ofthe T cell; and (ii) instructions for use of the compound to treat acondition associated with an excessive or unwanted T cell mediatedimmune response or excessive or unwanted T cell proliferation such asinflammation, autoimmunity, transplant rejection, an allergic condition,or a T cell cancer.

[0042] An advantage of the invention is that it can induce the depletionof T cells and/or the induction of apoptosis in T cells without causingan associated unwanted or harmful immune response. For example, in someembodiments the administration to an individual of an anti-PSGL-1antibody or a multimeric compound described herein does not result in anunwanted elevation in the levels of inflammatory cytokines such as IL-2or TNF-alpha.

[0043] Another advantage of the invention is that it causes thedepletion of T cells by the use of agonistic compositions that induceapoptosis of T cells. Accordingly, the invention provides for activeimmunosuppressive methods rather than passive immunosuppression thatresults from using antagonistic compositions (e.g., antagonisticanti-PSGL-1 antibodies or antagonistic soluble selectin fragments) thatact by binding immune receptors and preventing immune activationmediated by such receptors.

[0044] Another advantage of the invention is that it allows for thetargeting of a cell surface protein, PSGL-1, whose expression is largelylimited to leukocytes, and in particular T cells and NK cells.Therefore, the compounds described herein generally do not inducesignificant levels of apoptosis of other cell types such as liver cells.The targeting of T cells and NK cells (an important CD3⁻ cell typeinvolved in transplantation rejection) for selective depletion, withoutsignificantly inducing life-threatening systemic cytokine responses anddamaging other organ systems, is a desired characteristic of animmunosuppressive agent.

[0045] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by those of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, suitable methods and materialsare described below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of a conflict in terminology, the presentspecification will control. In addition, the described materials andmethods are illustrative only and are not intended to be limiting.

[0046] Other features and advantages of the invention will be apparentfrom the following detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047]FIG. 1 depicts the results of a time-course experiment thatinvestigated when activated T cells acquire sensitivity to TAB4 (ananti-PSGL-1 monoclonal antibody)-mediated apoptotic signals.

[0048]FIG. 2 depicts the results of cell surface biotinylation andimmunoprecipitation of the antigen recognized by the TAB4 antibody.

[0049]FIG. 3 depicts the expression of the PSGL-1 antigen on spleen CD4+T cells, CD8+ T cells, CD19+ B cells, and NK cells.

[0050]FIG. 4 depicts the expression of the PSGL-1 antigen on CD4⁺, CD8⁺,and CD4⁺8⁺, and CD4⁻8⁻ thymocytes.

[0051]FIG. 5 depicts the levels of IL-2 produced in mixed lymphocyteculture using spleen cells isolated from TAB4 (or hamster Ig)-treatedBalb/c mice as the responders and H2-mismatched C3H spleen cells as thestimulator.

[0052]FIG. 6 depicts western blot analyses demonstrating that (A)proteins immunoprecipitated with the TAB4 antibody can be recognized bya commercially available anti-PSGL-1 antibody and (B) preclearing of Tcell lysate with anti-PSGL-1 antibody can deplete the proteinsrecognized by the TAB4.

[0053]FIG. 7 depicts the percentage of surviving grafts in C57BL/6 micethat received a skin graft from Balb/c mice and were treated with ananti-PSGL-1 antibody (closed diamond) or a control antibody (opensquare).

[0054]FIG. 8 depicts the time course of the percentage of apoptotic Tcells following the treatment of activated human peripheral bloodmononuclear cells with an anti-human PSGL-1 antibody.

[0055]FIG. 9 depicts the incidence of diabetes in autoimmune non-obesediabetic (NOD) male mice that were treated with anti-PSGL-1 antibody(closed square) or a control antibody (open square).

[0056]FIG. 10 depicts the binding of mouse P-selectin, E-selectin, andL-selectin to mouse activated T cells.

[0057] FIGS. 11A-11C depict the induction of apoptosis of mouseactivated T cells by multimeric forms of E-selectin (FIG. 11A),P-selectin (FIG. 11B), and L-selectin (FIG. 1C).

[0058]FIG. 12 depicts the induction of apoptosis of mouse activated Tcells in vitro by the cross-linking of a soluble P-selectin-Fc fusionprotein.

DETAILED DESCRIPTION

[0059] The invention is directed to methods of modulating T cellactivity by modulating the function of PSGL-1 molecules residing on thesurface of a T cell. Engagement of PSGL-1 with agonist compositionsdescribed herein can cause the depletion of T cells and/or induce Tcells to undergo apoptosis. These agonist compositions are thereforeuseful as therapeutic agents for controlling immune-related conditionssuch as inflammatory diseases, autoimmune diseases, transplantrejection, allergic diseases, and/or T cell-derived cancers. The agonistcompositions are also useful in causing the depletion of T cells fromany biological sample where the presence or activity of T cells is notdesired.

[0060] PSGL-1 Protein

[0061] PSGL-1 is a cell surface adhesion molecule that is expressed onneutrophils, T and B-lymphocytes, NK cells, monocytes, dendritic cells,and primitive human CD34 hematopoietic progenitor cells. Through itsability to interact with selecting, PSGL-1 mediates the rolling ofleukocytes on the endothelium and the extravasation of leukocytes intoinflamed tissues. PSGL-1-mediated binding of T cells to E- andP-selectin, or migration, is differentially regulated. For instance, theappearance of CLA (cutaneous lymphocyte antigen) epitope is thought tobe induced on T cells undergoing naïve to memory transition. Onlyactivated helper 1 but not helper 2 T cells express functional PSGL-1and it capable of migration into the inflamed area of the skin.

[0062] PSGL-1 is a sialomucin that must be specifically sialylated,fucosylated, and sulfated to bind P-selectin. The PSGL-1 molecule existsin isoforms characterized by different degree of glycosylation andsulfation sites at their N-termini. Resting peripheral blood T and Bcells, lymphoid cell lines, and in vitro activated peripheral blood Tcells express similar level of PGSL-1. Yet, only activated T cellsdisplay a functional form of PSGL-1 and bind avidly to P-selectin. Suchactivation-dependent binding activity appears to be a result ofdifferential post-translational modification, as suggested by elevatedlevels of alpha (1,3) fucosyltransferases activities in activated Tcells. PSGL-1 isoforms also show differential affinity to L-selectin andE-selectin. For instance, human T cells exhibiting the CLA-positiveisoform can tether and roll on both E- and P-selectin, while T cellsexpressing PSGL-1 without the CLA epitope only bind to P-selectin.Furthermore, binding of PSGL-1 to P-selectin is contingent upon thepresence of the terminal decapeptide that contains three tyrosineresidues for sulfation and one threonine residue for glycosylation.

[0063] A PSGL-1 protein can be prepared by recombinant methods and/or byisolating a native PSGL-1 protein from biological material. Arecombinant PSGL-1 protein can be produced in prokaryotic or eukaryoticcells, either in vitro or in vivo. Nucleic acids encoding PSGL-1 can beused for recombinant production of the protein (see, e.g., GenBank™Accession NM_(—)003006 for an example of a nucleic acid encoding aPSGL-1 polypeptide). Antibodies directed to PSGL-1 are also well knownand can be used for purification of the antigen (see, e.g., Herron etal. (2000) Science Jun 2;288(5471):1653-56; WO 00/25808) and/or used inmethods described herein. PSGL-1 is further described in referencesincluding but not limited to Sako et al. (1993) Cell 75:1179; Vachino etal. (1995) J. Biol. Chem. 270:21966; and Veldman et al. (1995) J. Biol.Chem. 270:16470.

[0064] For recombinant production of PSGL-1, the simultaneous expressionof both PSGL-1 and its modifying alpha (1,3) fucosyltransferase,Fuc-TVII, may be required for the functional expression of PSGL-1. Inaddition or alternatively, recombinant production of PSGL-1 may beaccompanied by co-transfection with a nucleic acid encoding PACE forremoving the propeptide and/or or a nucleic acid encoding tyrosinesulfotransferase.

[0065] An anti-PSGL-1 antibody can be used to isolate and purify aPSGL-1 antigen from biological material. Any cell type expressing aPSGL-1 protein, e.g., T cells derived from an individual or a T cellline, can be used as a source of the protein. Once purified, the proteincan be used in a variety of methods as described herein. For example,the purified PSGL-1 protein can be used in an in vitro screen ofmodulators of PSGL-1 function on T cells or as an immunogen to prepareantibodies directed against the protein.

[0066] Anti-PSGL-1 Antibodies

[0067] PSGL-1 polypeptides (or immunogenic fragments or analogs thereof)can be used to generate antibodies useful in the methods of theinvention. As described above, PSGL-1 polypeptides or peptide fragmentsthereof can be produced by recombinant techniques or synthesized usingsolid phase synthesis methods. The recombinant PSGL-1 polypeptides or apeptide fragment thereof can be used as an immunogen to produceanti-PSGL-1 antibodies. In addition, an anti-PSGL-1 antibody, such asthe TAB4 monoclonal antibody, can be used to purify a PSGL-1polypeptide, e.g., a PSGL-1 polypeptide in its natural conformation,which can then be used as an immunogen to produce additional anti-PSGL-1antibodies.

[0068] An antibody of the invention can be a monoclonal, polyclonal, orengineered antibody that specifically binds to a PSGL-1 polypeptide. Anantibody that “specifically binds” to a particular antigen, e.g., aPSGL-1 polypeptide, will not substantially recognize or bind to othermolecules in a sample. Thus, the invention also features methods foridentifying a test compound (e.g., an antibody) that binds to apolypeptide of the invention by contacting the polypeptide with a testcompound and determining whether the polypeptide binds to the testcompound (e.g., by direct detection of the binding, detection of acompetitor molecule which disrupts binding of the test compound to thepolypeptide, and/or detection of binding using an assay forapoptosis-inducing activity).

[0069] In general, PSGL-1 polypeptides can be coupled to a carrierprotein, such as KLH, mixed with an adjuvant, and injected into a hostmammal. Antibodies produced in that animal can then be purified bypeptide antigen affinity chromatography.

[0070] In particular, various host animals can be immunized by injectionwith a PSGL-1 polypeptide or an antigenic fragment thereof. Commonlyemployed host animals include rabbits, mice, guinea pigs, and rats.Various adjuvants that can be used to increase the immunologicalresponse depend on the host species and include Freund's adjuvant(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Potentially useful human adjuvants include BCG (bacilleCalmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies areheterogeneous populations of antibody molecules that are contained inthe sera of the immunized animals.

[0071] Antibodies within the invention therefore include polyclonalantibodies and, in addition, monoclonal antibodies, humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab′)2fragments, and molecules produced using a Fab expression library.

[0072] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be prepared using the PSGL-1polypeptides described above and standard hybridoma technology (see, forexample, Kohler et al., Nature 256:495 [1975]; Kohler et al., Eur JImmunol 6:511 [1976]; Kohler et al., Eur J Immunol 6:292 [1976];Hammerling et al., Monoclonal Antibodies and T Cell Hybridomas,Elsevier, N.Y. [1981]).

[0073] In particular, monoclonal antibodies can be obtained by anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture such as described in Kohler et al.,Nature 256:495 (1975), and U.S. Pat. No. 4,376,110; the human B-cellhybridoma technique (Kosbor et al., Immunology Today 4:72 [1983]; Coleet al., Proc Natl Acad Sci USA 80:2026 [1983]), and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96 [1983]). Such antibodies can be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. The hybridoma producing the mAb of this invention may becultivated in vitro or in vivo. The ability to produce high titers ofmAbs in vivo makes this a particularly useful method of production.

[0074] Once produced, polyclonal or monoclonal antibodies are tested forspecific PSGL-1 recognition by Western blot or immunoprecipitationanalysis by standard methods, for example, as described in Ausubel etal., supra. Antibodies that specifically recognize and bind to PSGL-1are useful in the invention. Anti-PSGL-1 antibodies that bind to thePSGL-1 antigen on the surface of a T cell, e.g., a CD3+ cell, and inducethe depletion and/or apoptosis of T cells in an individual areparticularly useful.

[0075] The antibodies can be used, for example, as part of a therapeuticregime (e.g., to reduce or eliminate an undesirable immune response,such as a T cell mediated immune response, associated with conditionssuch as inflammatory diseases, autoimmune diseases, transplantrejection, allergic diseases, and T cell-derived cancers). Antibodiesalso can be used in a screening assay to measure the ability of acandidate compound to bind to PSGL-1.

[0076] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc Natl Acad Sci USA 81:6851 [1984];Neuberger et al., Nature 312:604 [1984]; Takeda et al., Nature 314:452[1984]) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region.

[0077] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692)can be adapted to produce single chain antibodies against a PSGL-1polypeptide, or a fragment thereof. Single chain antibodies are formedby linking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

[0078] Antibody fragments that recognize and bind to specific epitopescan be generated by known techniques. For example, such fragmentsinclude but are not limited to F(ab′)2 fragments that can be produced bypepsin digestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)2 fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science 246:1275 [1989]) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0079] Antibodies can be humanized by methods known in the art. Forexample, monoclonal antibodies with a desired binding specificity can becommercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto,Calif.). Fully human antibodies, such as those expressed in transgenicanimals are also features of the invention (Green et al., NatureGenetics 7:13 [1994]; and U.S. Pat. Nos. 5,545,806 and 5,569,825).

[0080] Multimeric Compounds

[0081] Multimeric compounds that bind to a plurality of PSGL-1 proteinson the surface of a T cell or NK cell can be used to induce apoptosis inthe cell. The multimeric compound contains at least two polypeptidechains. Each of the polypeptide chains contains (i) a binding domainthat binds to PSGL-1, and (ii) a heterologous amino acid sequence.

[0082] In general, a multimeric compound binds to at least two differentPSGL-1 proteins on the surface of a given cell. However, a multimericcompound can be formulated to have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, ormore distinct PSGL-1 binding domains, thereby causing the multimericcompound to bind to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more differentPSGL-1 proteins on the surface of a given cell.

[0083] A binding domain can contain any amino acid sequence (or anyamino acid sequence with a modification such as, e.g., glycosylationand/or sulfation) that binds to PSGL-1. The binding domain cancorrespond to either a naturally occurring or a non-naturally occurringamino acid sequence. For example, a binding domain can contain thePSGL-1-binding domain of a selectin (e.g., P-selectin, E-selectin, orL-selectin). A polypeptide containing a PSGL-1 binding domain of aselectin can optionally include: (i) an extracellular domain of theselectin (e.g., P-selectin, E-selectin, or L-selectin); (ii) a calciumdependent lectin domain of the selectin (e.g., P-selectin, E-selectin,or L-selectin); or (iii) a fragment of the extracellular domain of theselectin (e.g., P-selectin, E-selectin, or L-selectin) that mediatesbinding to PSGL-1. In addition to these naturally occurring amino acidsequences, one or more amino acid changes can be introduced into anaturally occurring PSGL-1 binding domain, resulting in a non-naturallyoccurring sequence that retains PSGL-1-binding function. For example, apolypeptide can contain an amino acid sequence that binds to PSGL-1 andis at least 80%, 85%, 90%, 95%, or 98% identical to any of: (i) anextracellular domain of a selectin (e.g., P-selectin, E-selectin, orL-selectin); (ii) a calcium dependent lectin domain of a selectin (e.g.,P-selectin, E-selectin, or L-selectin); or (iii) a fragment of theextracellular domain of a selectin (e.g., P-selectin, E-selectin, orL-selectin) that mediates binding to PSGL-1. Standard molecular biologymutagenesis techniques can be used to introduce changes into a nucleicacid sequence encoding a PSGL-1 binding domain. Modified binding domainscan then be tested for their ability to bind to PSGL-1, e.g.,immobilized PSGL-1 or PSGL-1 on the surface of a cell. A binding domaincan also contain the PSGL-1 binding domain of an anti-PSGL-1 antibody ora polypeptide selected from a phage display library, or a an amino acidsequence that binds to PSGL-1 and is at least 80%, 85%, 90%, 95%, or 98%identical to the PSGL-1 binding domain of an anti-PSGL-1 antibody or apolypeptide selected from a phage display library.

[0084] A PSGL-1-binding domain can contain an amino acid sequence thatcorresponds to a PSGL-1-binding fragment of P-selectin. An example of apolypeptide chain (of a multimeric compound described herein) containingsuch an amino acid sequence is a recombinant mouse P-selectin/Fc chimera(available from R&D Systems, Minneapolis, Minn.) containing thefollowing components: (i) CD33 signal peptide (Met1-Ala16); (ii) mouseP-selectin (Trp42-Ala709 of extracellular domain); (iii) IEGRMD (SEQ IDNO:1); and (iv) Human IgG1 (Pro100-Lys330). A second example of apolypeptide chain containing such an amino acid sequence is arecombinant human P-selectin/Fc chimera (available from R&D Systems,Minneapolis, Minn.) containing the following components: (i) humanP-selectin (Met1-Ala771, extracellular domain); (ii) IEGRMD (SEQ ID NO:1); and (iii) Human IgG1 (Pro100-Lys330).

[0085] A PSGL-1-binding domain can contain an amino acid sequence thatcorresponds to a PSGL-1-binding fragment of E-selectin. An example of apolypeptide chain (of a multimeric compound described herein) containingsuch an amino acid sequence is a recombinant mouse E-selectin/Fc chimera(available from R&D Systems, Minneapolis, Minn.) containing thefollowing components: (i) mouse E-selectin (Met1-Pro557, extracellulardomain); (ii) IEGRMD (SEQ ID NO:1); (iii) Human IgG1 (Pro100-Lys330);and (iv) HHHHHH (SEQ ID NO:2). A second example of a polypeptide chaincontaining such an amino acid sequence is a recombinant humanE-selectin/Fc chimera (available from R&D Systems, Minneapolis, Minn.)containing the following components: (i) human E-selectin (Met1-Pro556,extracellular domain); (ii) IEGRMD (SEQ ID NO:2); (iii) human IgG1(Pro100-Lys330); and (iv) HHHHHH (SEQ ID NO:2).

[0086] A PSGL-1 binding domain can contain an amino acid sequence thatcorresponds to a PSGL-1-binding fragment of L-selectin. An example of apolypeptide chain (of a multimeric compound described herein) containingsuch an amino acid sequence is a recombinant mouse L-selectin/Fc chimera(available from R&D Systems, Minneapolis, Minn.) containing thefollowing components: (i) mouse L-selectin (Met1-Asn332, extracellulardomain); (ii) IEGRMD (SEQ ID NO: 1); (iii) Human IgG1 (Pro100-Lys330);and (iv) HHHHHH (SEQ ID NO:2). A second example of a polypeptide chaincontaining such an amino acid sequence is a recombinant humanL-selectin/Fc chimera (available from R&D Systems, Minneapolis, Minn.)containing the following components: (i) human L-selectin (Met1-Asn332,extracellular domain); (ii) IEGRMD (SEQ ID NO:1); (iii) Human IgG1(Pro100-Lys330); and (iv) HHHHHH (SEQ ID NO:2).

[0087] A multimeric compound can be formulated as a homo-multimericcompound or a hetero-multimeric compound. A homo-multimeric compoundcontains only polypeptide chains that have identical PSGL-1 bindingdomains. For example, a homo-multimeric compound can contain polypeptidechains containing identical PSGL-1-binding fragments of P-selectin. Ahetero-multimeric compound contains polypeptide chains that havedifferent PSGL-1 binding domains. For example, a hetero-multimericcompound can contain a first polypeptide chain that contains aPSGL-1-binding fragment of P-selectin and a second polypeptide chainthat contains a PSGL-1-binding fragment of E-selectin.

[0088] A heterologous amino acid sequence can be any amino acidsequence. However, the amino acid sequence of the polypeptide chainsdescribed herein does not correspond to the sequence of a naturallyoccurring protein. A heterologous amino acid sequence contains one ormore amino acids that permit the linkage of the polypeptide chains. Forexample, the one or more amino acids can covalently link, e.g., via adisulfide linkage, the polypeptide chains. One example of a heterologoussequence is an immunoglobulin heavy chain constant region. Disulfidebonding between Fc regions of two polypeptide chains can result in theformation of a dimeric compound.

[0089] In addition to contributing to the linkage of the polypeptidechains, the heterologous amino acid sequence can also contain across-linker binding region, e.g., a cell surface receptor bindingregion. Upon the binding of an agent to such a binding region,cross-linking of the polypeptide chains and the cell surface PSGL-1proteins to which they are bound can result. An immunoglobulin heavychain constant region contains an Fc receptor binding region. Across-linker can be, for example, an antibody (e.g., an anti-Fcantibody) that specifically binds to the cross-linker binding region ofthe heterologous amino acid sequence.

[0090] Screening Assays for Compounds that Modulate PSGL-1 Function

[0091] The invention also encompasses methods for identifying compoundsthat interact with PSGL-1 (or a domain of PSGL-1) including, but notlimited to, compounds that induce T cell depletion and/or T cellapoptosis upon binding to PSGL-1. Also included are compounds thatmodulate the interaction of PSGL-1 with transmembrane, extracellular, orintracellular proteins that regulate PSGL-1 activity and compounds whichmodulate PSGL-1 activity.

[0092] The compounds that may be screened in accordance with theinvention include, but are not limited to peptides, antibodies andfragments thereof, and other organic compounds that bind to PSGL-1 andmodulate a biological function mediated by PSGL-1, as described herein.

[0093] Such compounds may include, but are not limited to, peptides suchas, for example, soluble peptides, including but not limited to membersof random peptide libraries; (Lam et al., Nature 354:82 [1991]; Houghtenet al., Nature 354:84 [1991]), and combinatorial chemistry-derivedmolecular library made of D- and/or L configuration amino acids,phosphopeptides (including, but not limited to, members of random orpartially degenerate, directed phosphopeptide libraries; Songyang etal., Cell 72:767 [1993]), antibodies (including, but not limited to,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or singlechain antibodies, and FAb, F(ab′)2 and FAb expression library fragments,and epitope-binding fragments thereof), and small organic or inorganicmolecules.

[0094] Other compounds which can be screened in accordance with theinvention include but are not limited to small organic molecules thataffect an activity of the PSGL-1 protein, as described herein.

[0095] Computer modeling and searching technologies permitidentification of compounds, or the improvement of already identifiedcompounds, that can modulate PSGL-1 expression or activity. Havingidentified such a compound or composition, the active sites or regionsare identified. Such active sites might typically be a binding site fora natural modulator of activity. The active site can be identified usingmethods known in the art including, for example, from the amino acidsequences of peptides, from the nucleotide sequences of nucleic acids,or from study of complexes of the relevant compound or composition withits natural ligand. In the latter case, chemical or X-raycrystallographic methods can be used to find the active site by findingwhere on the factor the modulator (or ligand) is found.

[0096] Although described above with reference to design and generationof compounds which could alter binding, one could also screen librariesof known compounds, including natural products or synthetic chemicals,and biologically active materials, including proteins, for compoundswhich bind to a PSGL-1 protein and cause T cell depletion and/or induceT cell apoptosis.

[0097] In vitro systems may be designed to identify compounds capable ofinteracting with PSGL-1 (or a domain of PSGL-1). Compounds identifiedmay be useful, for example, in modulating T cell activity as describedherein and thus may be useful for the treatment of conditions associatedwith an excessive or unwanted T cell mediated immune response orexcessive or unwanted T cell proliferation such as inflammation,autoimmunity, transplant rejection, an allergic condition, or a T cellcancer.

[0098] The principle of the assays used to identify compounds that bindto PSGL-1 involves preparing a reaction mixture of PSGL-1 (or a domainthereof) and the test compound under conditions and for a timesufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. The PSGL-1 species used can vary depending upon the goal of thescreening assay. In some situations it is preferable to employ a peptidecorresponding to a domain of PSGL-1 fused to a heterologous protein orpolypeptide that affords advantages in the assay system (e.g., labeling,isolation of the resulting complex, etc.) can be utilized.

[0099] The screening assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay involves anchoring PSGL-1protein, polypeptide, peptide or fusion protein or the test substanceonto a solid phase and detecting PSGL-1/test compound complexes anchoredon the solid phase at the end of the reaction. In one embodiment of sucha method, the PSGL-1 reactant may be anchored onto a solid surface, andthe test compound, which is not anchored, may be labeled, eitherdirectly or indirectly.

[0100] In practice, microtiter plates may conveniently be utilized asthe solid phase. The anchored component may be immobilized bynon-covalent or covalent attachments. Non-covalent attachment may beaccomplished by simply coating the solid surface with a solution of theprotein and drying. Alternatively, an immobilized antibody, preferably amonoclonal antibody, specific for the protein to be immobilized may beused to anchor the protein to the solid surface. The surfaces may beprepared in advance and stored.

[0101] In order to conduct the assay, the nonimmobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously non-immobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the previously non-immobilized component is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the previouslynon-immobilized component (the antibody, in turn, may be directlylabeled or indirectly labeled with a labeled anti-Ig antibody).

[0102] Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for PSGL-1protein, polypeptide, peptide or fusion protein or the test compound toanchor any complexes formed in solution, and a labeled antibody specificfor the other component of the possible complex to detect anchoredcomplexes.

[0103] Alternatively, cell-based assays can be used to identifycompounds that interact with PSGL-1. To this end, cell lines thatexpress PSGL-1, or cell lines that have been genetically engineered toexpress PSGL-1 can be used. Cell based assays are particularly usefulfor evaluating the functional effects of a compound identified by ascreen described herein. For example, once a compound is identifiedbased upon its ability to bind to a PSGL-1 protein, the compound canthen be tested for its ability to, e.g., induce T cell apoptosis invitro or in vivo or deplete T cells in vitro or in vivo.

[0104] Pharmaceutical Compositions

[0105] Given that an object of the present invention is to alter animmune response in an individual, a pharmaceutical compositioncontaining, for example, antibodies, multimeric compounds, smallmolecules, or other compounds that specifically bind PSGL-1 polypeptidesare also a feature of the invention. In a preferred example, thecompound functions as an agonist of PSGL-1.

[0106] Pharmaceutical compositions for use in accordance with thepresent invention can be formulated in a conventional manner using oneor more physiologically acceptable carriers or excipients. Thus, thecompounds and their physiologically acceptable salts and solvates may beformulated for administration by a variety of routes of administration.

[0107] The compounds may be formulated for parenteral administration byinjection, for example, by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, for example, sterile pyrogen-freewater, before use.

[0108] Methods of Controlling a T Cell-Mediated Immune Response andDepleting T Cell Populations

[0109] Compounds such as those detailed in the screening assaysdescribed herein may be useful, for example, in modulating a biologicalfunction mediated by a PSGL-1 polypeptide and/or for the treatment ofdisorders associated an excessive or unwanted immune response, e.g., a Tcell-mediated immune response. These compounds include, but are notlimited to peptides, antibodies and fragments thereof, and other organiccompounds that bind to PSGL-1 on the surface of a T cell and induce asignal transduction pathway that results in the death of the T cell. Themethods of the invention optionally include the addition of across-linking agent that induces the cross-linking of PSGL-1 on thesurface of a cell. The compounds described herein can be used in anyinstance wherein the depletion or elimination of T cell activity isdesired. Particularly useful conditions that can be treated with thecompounds of the invention include inflammatory diseases, autoimmunediseases, transplant rejection, allergic diseases, and T cell-derivedcancers.

[0110] Examples of conditions that can be treated with the anti-PSGL-1compounds described herein include, but are not limited to, diabetesmellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoidarthritis, osteoarthritis, and psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjögren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, type Idiabetes, inflammatory bowel diseases, ulcerative colitis, asthma,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, drug eruptions, leprosy reversal reactions, erythema nodosumleprosum, autoimmune uveitis, allergic encephalomyelitis, acutenecrotizing hemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis,uveitis posterior, interstitial lung fibrosis, graft-versus-hostdisease, cases of transplantation (including transplantation usingallogeneic or xenogeneic tissues) such as bone marrow transplantation,liver transplantation, or the transplantation of any organ or tissue,allergies such as atopic allergy, AIDS, and T-cell neoplasms such asleukemias and/or lymphomas.

[0111] The methods of the invention can be used to deplete T cells froma cell population, either in vitro or in vivo. For example, a biologicalsample derived from an individual can be depleted of T cells in vitro bycontacting the sample with an anti-PSGL-1 compound described herein,optionally together with a cross-linking agent. This method can beuseful, e.g., by allowing for the enrichment of non-T cells in a cellpopulation as well as by reducing or eliminating T cell activity from acell population.

[0112] The following are examples of the practice of the invention. Theyare not to be construed as limiting the scope of the invention in anyway.

EXAMPLES Example 1 Preparation of an Anti-T Cell Apoptosis InducingProtein (“TAIP”) Monoclonal Antibody

[0113] A TAIP-specific monoclonal antibody was generated by applying thewell known cell fusion methods of Kohler and Milstein ((1976) EuropeanJournal of Immunology 6:511-519) to produce a hybridoma secretingdesired antibodies. Antibody-producing cells from a hamster injectedwith Concanovalin A (Con A)-activated Balb/c spleen T cells were fusedwith a myeloma cell line to form an antibody secreting hybridoma. Thetwo populations of cells were fused with polyethylene glycol, and theresulting antibody producing cells were cloned and propagated bystandard tissue culture methods. One hybridoma generated according tothese methods secreted a monoclonal antibody, designated TAB4, that wasable to induce T cell apoptosis in vitro and deplete T cells in vivo.The protein recognized by TAB4 was designated T cell apoptosis inducingprotein (TAIP).

[0114] C57BL/6J (B6) and BALB/c mice were purchased from the Jackson lab(Bar Harbor, Me.). Syrian hamsters were purchased from the Animal CoreFacility, National Taiwan University Medical College.

[0115] Concentrated culture supernatant of the TAB4 hybridoma was spunat 20,000×g for 10 minutes and the supernatant was diluted at a 1:1ratio with the binding buffer (0.1 M sodium acetate, pH 5.0). A proteinG column (approximately 1 ml bed volume) was washed three times with 3-5ml of the binding buffer. The cleared culture supernatant was loaded tothe protein G column and the flow-through was collected and reloaded tothe column. The column was washed with 6-10 ml of the binding buffer andthe bound antibody was eluted from the column with 5 ml of the elutionbuffer (0.1 M glycine-HCl, pH 2.8). Each fraction contained 1 ml of theeluted antibody and the eluted fraction was adjusted to neutral pH bymixing each 1 ml fraction with 50 microliters of 1 M Tris-HCl, pH 7.5.Fractions containing the antibody were pooled and dialyzed against 2liters of PBS, pH 7.4 three times at three hours for each dialysis.Protein concentration in the antibody samples were determined with theprocedure described by Bradford using the Bio-Rad Protein Assay(BIO-RAD, Hercules, Calif.).

Example 2 Preparation of a Mouse Spleen Cell Suspension and theActivation and Enrichment of T cells

[0116] Mouse spleen was immersed in 8 ml of Hank's balanced saltsolution (HBSS), gently minced with a sterile cover slip, transferred toa 15 ml centrifuge tube (Costar), and spun at 200×g for 5 minutes. Thesupernatant was discarded and the cell pellet was resuspended in theresidual buffer by gently tapping the wall. The contaminating red bloodcells (RBC) were lysed by the addition of 1 ml of RBC lysis buffer (0.6M NH₄Cl, 0.17 M Tris-base, pH 7.65), followed by a 2 min incubation atroom temperature and rapid quenching with 9 ml of HBSS. The cells werepelleted at 200×g for 5 minutes, washed twice and resuspended in RPMImedium. The concentration and viability of cells in the mixture weredetermined with a hemocytometer (Cambridge Scientific Inc.) and Trypanblue exclusion.

[0117] The spleen cells were adjusted to a final concentration of3×10⁶/ml with RPMI medium and Concanovalin A was added to a finalconcentration of 2 micrograms/ml to activate the T cells. The cellsuspension was transferred to a 6-well culture plate (5 ml/well) or a10-cm culture dish (10 ml/dish) and incubated at 37° C., 5% CO₂ for 48hours before harvesting. The activated spleen cells, including activatedT cells, were resuspended in 5 ml of HBSS and carefully overlaid on topof a 5 ml 55% cushion of Percoll solution in a centrifuge tube. Care wastaken not to disturb the separated layers. The cells were spun at1,900×g for 13 minutes at 25° C. without the brake. The enriched T cellswere collected from the interface of the two layers, washed twice withHBSS, and were ready for experiments.

Example 3 Apoptosis of Activated T cells

[0118] Activated T cells (see Example 2) were resuspended to a finalconcentration of 5×10⁵ cells/ml in RPMI medium containing 5 ng/ml ofIL-2, and treated with control Ig, TAB4, or anti-CD3 according to theconditions shown in Table 1. TABLE 1 Experiment groups Treatment*Negative control 3 ug/ml hamster Ig 5 ng/ml IL-2 3 ug/ml cross-linkerantibody (anti-hamster Ig) TAB4 3 ug/ml TAB4 hamster mAb 5 ng/ml IL-2 3ug/ml cross-linker antibody (anti-hamster Ig) Positive control 1 ug/mlanti-CD3 mAb 5 ng/ml IL-2 1 ug/ml cross-linker antibody (anti-mouse Ig)

[0119] After an incubation period of 18-24 hours, the extent ofapoptosis in each culture was determined using the 7-AAD apoptosisassay. The treated cells were transferred to FACS tubes (Falcon), washedtwice with ice-cold FACS solution (1% fetal bovine serum, 0.05% sodiumazide in PBS), pelleted at 200×g at 4° C. The cells were resuspended inice-cold FACS solution to a final concentration of 1-2×10⁷ cells/ml. Forstaining, 0.1 ml of the resuspended cells were mixed with 7-AAD to afinal concentration of 2 ug/ml and then incubated at 4° C. in the darkfor 20 minutes. Finally, the stained cells were washed twice withice-cold FACS solution, resuspended in 0.5 ml of FACS solution andanalyzed with BD LSR flow cytometer (Beckton Dickison).

[0120]FIG. 1 depicts the results of a representative time-courseexperiment that investigated when activated T cells acquire sensitivityto TAB4 (anti-TAIP)-mediated apoptotic signals. Mouse splenocytes wereactivated with Con-A and maintained in IL-2 containing medium. ActivatedT cells were harvested, resuspended, and challenged with TAB4 monoclonalantibody or control hamster IgG in the presence of anti-hamster IgGantibody as cross-linker. The ability of TAIP cross-linking to inducelow level (6.5%) of apoptotic cell death was evident on day one.However, the extent of TAB4-induced apoptosis increased from 17% on day2, peaked at 52% on day 4, and declined to 44% on day 6. The controlhamster IgG did not induce specific apoptotic T cell death, as comparedwith the cultures that received only IL-2. Anti-CD3 (as positivecontrol) induced apoptosis in 38% of T cell after 48 hours of activation(data not shown).

Example 4 Expression of the TAIP Antigen in Different Tissues

[0121] Cells were washed twice with ice-cold FACS solution (1% fetalbovine serum, 0.05% sodium azide in PBS) and spun at 200×g at 4° C. in aFACS tube (Falcon). The cells were resuspended in ice-cold FACS solutionto a final concentration of 1×10⁷ cells/ml and a 0.1 ml aliquot of theresuspended cells in a FACS tube (Falcon) was used for each assay. Forsurface staining, the TAB4 monoclonal antibody or a control hamster Igat a final concentration of 2 ug/ml were added to the cells and themixtures were incubated at 4° C. for 30 minutes in the dark. The cellswere washed once with ice-cold FACS and then stained with: (1) forspleen cells, cychrome-conjugated anti-CD3 antibody (2 ug/ml),FITC-conjugated anti-hamster Ig, and PE-conjugatedanti-CD8/CD4/CD19/CD11b/pan-NK/I-A/I-E/Mac-3 antibody (2 ug/ml) in 100ul of ice-cold FACS solution; and (2) for thymus cells, FITC-conjugatedanti-hamster Ig, PE-conjugated anti-CD8, and cychrome-conjugatedanti-CD4 antibodies (2 ug/ml) in 100 ul of ice-cold FACS solution. Thereaction was performed at 4° C. for 30 minutes in the dark. Finally, thestained cells were washed twice with ice-cold FACS solution, resuspendedin 1 ml of FACS solution and analyzed with BD LSR flow cytometer(Beckton Dickison).

[0122]FIGS. 3 and 4 demonstrate by FACS analysis the distribution ofTAIP antigen on the various subpopulations of splenocytes andthymocytes. As shown in FIG. 3, CD19⁺ B cells expressed low butdetectable amounts of TAIP proteins on the surface. Significantly higheramounts of TAIP proteins were detected on CD3⁺ T cells and a fraction ofNK cells. Most of the CD4⁺, CD8⁺, and CD4⁺8⁺ thymus T cells expressedsignificant amounts of TAIP proteins. In contrast, the TAIP proteinswere expressed only on a small population of CD4⁻8⁻ thymus T cells (FIG.4).

[0123] Tissues from B6 and BALB/c mice, including brain, thymus, heart,lung, liver, stomach, kidney, spleen, and skin, were collected, fixed in10% formaldehyde overnight at room temperature, and embedded in paraffinblocks. Tissue sections, at a 4 um thickness, were prepared from theparaffin block with Leica RM2135 microtome, spread in 45° C. water, andlaid on a coated slide. The slides were dried in 37° C. and were readyfor subsequent experiments.

[0124] Slides containing the tissue paraffin sections were dewaxed anddried through a xylenes-100% ethanol series according to standardprotocol and were finally kept in 100% ethanol. The sections wererehydrated through a 100% ethanol-90% ethanol-85% ethanol-70%ethanol-PBS serial incubation according to standard protocol to a finalPBS solution. The following reactions were all performed in a humidifiedbox. Non-specific binding were blocked by incubating the tissue sectionsin blocking buffer (1% normal goat serum) for 1 hour at room temperature(or 4° C. overnight). The blocking buffer was removed and TAB4 or normalhamster Ig (1:200 dilution) was added to the sections and incubationcontinued for another hour at room temperature (or 4° C. overnight). Thesections were washed twice in PBS, for 5 minutes each, to remove theprimary antibody, reacted with 1:250 diluted alkalinephosphatase-conjugated goat anti-hamster Ig, and incubated at roomtemperature for 1 hour. The sections were again washed twice with PBS, 5minutes each, to remove the antibody-enzyme conjugate and the colorreaction was developed with BCIP/NBT substrate solution at roomtemperature for 30 minutes in the dark. The sections were washed againwith PBS to remove excess enzyme substrate, dehydrated through thePBS-ethanol-xylenes series, and mounted for microscopy.

[0125] The results indicated that the TAIP proteins expression weredetected only in bone marrow derived-tissues but not on the rest of thetissues tested.

Example 5 Cell Surface Biotinylation and Immunoprecipitation of the TAIPAntigen

[0126] 5×10⁷ RL♂1 or NIH-3 T3 cells were surface biotinylated in 1 ml ofPBS containing 0.5 mg/ml Sulfo-NHS-biotin (Pierce) for 30 minutes onice. The reaction was terminated by incubating the cells with 0.5 ml ofDulbecco's modified Eagle's medium (Life Technologies, Inc.) for 10minutes on ice. Cells were washed with 1 ml of Dulbecco's modifiedEagle's medium once and with 1 ml of phosphate-buffered saline twice.

[0127] Labeled cells were lysed at a density of 5.0×10⁷ cells/ml in coldlysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 160 mM NaCl, 1 mMCaCl₂) containing complete protease inhibitor cocktail (Roche) for 15minutes, and insoluble material was pelleted at 10,000×g for 10 minutes;these and all subsequent steps were performed at 4° C. Forimmunoprecipitation, the lysate was preincubated for 30 minutes with 50μl of packed protein G-Sepharose (Amersham Pharmacia Biotech) to removenon-specifically binding proteins. Beads were pelleted, and aliquots ofthe supernatant (routinely corresponding to 5.0×10⁷ cells) wereincubated with 20 μl of protein G-Sepharose preloaded with 10 μg of mAbTAB4 or IgG from normal hamster serum. After incubation for 4 h at 4°C., the resin was washed four times with washing buffer (0.05% TritonX-100, 50 mM Tris-HCl, pH 8.5, 400 mM NaCl, 1 mM CaCl₂, 1 mg/mlovalbumin), twice with a similar washing buffer, containing 250 mMinstead of 400 mM NaCl. Proteins specifically bound to the TAB4 wereeluted with 50 μl of 1×SDS sample buffer. Eluted proteins were separatedby 8% SDS-PAGE and transferred to nitrocellulose membrane (Millipore).Filters were analyzed for biotinylated proteins withperoxidase-conjugated Avidin (PharMingen) and developed with theChemiluminescence reagent (NEN™ Life Science Products).

[0128] As shown in FIG. 2, a biotinylated surface protein with amolecular weight of approximately 120-kD was identified by TAB4 in RL.1cells (TAIP⁺ T cells), but not in 3T3 cells (TAIP⁻ cells). In contrast,protein G sepharose coated with hamster normal serum could not retrievethis 120-kDa protein. These results suggest that this 120-kDa protein isthe antigen recognized by monoclonal antibody TAB4 on the cell surfaceof T cells.

Example 6 Depletion of T Cells In Vivo

[0129] To examine the effects of TAB4 on populations of T cells andother cells in vivo, mice were injected with 300 ug of TAB4 or controlhamster Ig intraperitoneally and, on day 4, splenocytes, thymocytes, andperipheral blood mononuclear cells were harvested for the total cellcount and for the analyses of cell surface markers by FACS.

[0130] For FACS assays, the cells were fixed with 2% paraformaldehyde at4° C. for 20 minutes, washed twice, and resuspended in ice-cold FACSsolution to a final concentration of 1×10⁷ cells/ml. A 100 ul aliquot ofthe resuspended cells in a FACS tube (Falcon) was used for each assay.TAB4 or control hamster Ig at a final concentration of 2 ug/ml wereadded to the cells and the mixtures were incubated at 4° C. for 30minutes in the dark. The cells were washed once with ice-cold FACS andreacted with: (1) for spleen cells, cychrome-conjugated anti-CD3antibody (2 ug/ml), FITC-conjugated anti-hamster Ig and PE-conjugatedanti-CD8/CD4/CD19/CD11b/pan-NK/I-A/I-E/Mac-3 antibody (2 ug/ml) in 100ul of ice-cold FACS solution; and (2) for thymus cells, FITC-conjugatedanti-hamster Ig, PE-conjugated anti-CD8, and cychrome-conjugatedanti-CD4 antibodies (2 ug/ml) in 100 ul of ice-cold FACS solution. Thereaction was performed at 4° C. for 30 minutes in the dark. Finally, thestained cells were washed twice with ice-cold FACS solution, resuspendedin 1,000 ul of FACS solution and analyzed with BD LSR flow cytometer(Beckton Dickison).

[0131] Four days after the injection, the percentages of CD3⁺ T cells inperipheral blood leukocytes (PBL) decreased from 36.7% in control miceto 4.1% in TAB4-treated mice (Table 2). TAB4 treatment caused a slightreduction in the total number of splenocytes. However, in TAB4 treatedmice, there was a 62% decrease in the number of CD3⁺ T cells, a 50%decrease in the number of NK cells, and a slightly increase in the totalnumber of CD19+ B cells. The total number of thymocytes recovered fromTAB4 treated mice was only 48% of the level seen in control (52%reduced). Moreover, except for CD4+ T cells, all other CD8+, CD4+CD8+,and CD4−CD8− T cells were reduced, with CD4+CD8+ subpopulation being themost profoundly affected (64.7% reduction). TABLE 2 Spleen No NormalTA-B4- Depletion ×10⁶ Treatment Hamster Ig treated (%) Total 123 93.3105 14.6 Splenocytes CD3⁺ T cells 32.8 28.4 12.4 62.2 CD3⁻ CD19⁺ 72.253.4 72.9 −0.8 CD3⁻ NK⁺ 3.6 2.4 1.80 50 Peripheral Blood Leukocytes NoNormal TA-B4- Depletion Treatment Hamster Ig treated (%) CD3⁺ T cells36.7% 36% 4.1% 88.8% Thymus No Normal TA-B4- Depletion ×10⁶ TreatmentHamster Ig treated (%) Total 94 229 45 52.1 Thymocytes CD4⁺ 9.3 28.410.9 −16.6 CD8⁺ 5.2 7.7 3.6 30.3 CD4⁺ CD8⁺ 73.8 182 26 64.7 CD4⁻ CD8⁻5.6 10.5 4.5 19.3

EXAMPLE 7 Anti-TAIP Antibody does not Induce IL-2 or TNF-alpha Secretion

[0132] Balb/c mice (H-2d) were intraperitoneally injected with 300micrograms of TAB4 or control hamster Ig. Splenocytes were isolated 7days after injection, and used as responders in culture with mitomycinC-treated C3H(H-2k) splenocytes (as stimulators). Three days later, theculture supernatants were harvested and the IL-2 content was measured byELISA set (PharMingen). As shown in FIG. 5, the IL-2 production wassuppressed in responder cells derived from TAB4-treated mice as comparedwith that of control mice. The plasma levels of IL-2 and TNF-alpha werealso analyzed and no significant difference was noted in the levels ofIL-2 (or TNF-alpha) in the sera of the control and the TAB4 treatedmice. Since production of IL-2 is central to the activity of T cells,the results show that a TAIP-specific antibody, such as TAB4, can beused in vivo to manipulate T cells and control unwanted T cell-mediatedimmune responses such as those associated with autoimmune diseases andtransplantation rejection.

EXAMPLE 8 Use of an Anti-TAIP Antibody to Prevent Transplant Rejection

[0133] Mice (obtained from Jackson Laboratory) at 8 to 12 weeks of agewere anesthetized with Acepromazin maleate (Fermenta Animal Health Co.,Kansas City, Mo.). Prior to skin grafting, non-thymectomized recipientC57BL/6 mice (H-2^(b)) were injected intraperitoneally with 500 ug ofTAB4 or isotype control antibodies seven days before skin transplantsurgery. Seven days later, a lateral flank of skin from fully allogeneicmismatched Balb/cj mice (H-2^(d)) was grafted on the lateral flank ofthe antibody pre-treated C57BL/6 mice. Seven days post transplantation,the mice were again injected with 500 ug of TAB4 or isotype controlantibody. The mice were monitored every day after graft transplantation.The grafts were considered rejected when 50% donor skin was necrotic.The percent of graft survival is shown in FIG. 7 (n=8). The data showthat TAB4 antibody treatments prolonged the survival of the allogeneicskin grafts.

EXAMPLE 9 Identification of TAIP as PSGL-1

[0134] P-selectin glycoprotein ligand-1 (PSGL-1), also named CD162, isthe main P-selectin ligand expressed on leukocytes, including T cells(Sako et al. (1993) Cell 75:1179; Vachino et al. (1995) J. Biol. Chem.270:21966; Veldman et al. (1995) J. Biol. Chem. 270:16470). Biochemicalcharacteristics of TAIP, such as its molecular weight and its tendencyfor dimerization suggested the possibility that TAB4 may be analogous toPSGL-1. To investigate the relationship between these two antigens, thefollowing were tested: 1) whether the antigen precipitated by TAB4 canbe recognized by a commercially-available anti-PSGL1 antibody; and 2)whether an anti-PSGL1 antibody can deplete TAB4 from the cell lysate.

[0135] RL♂1 T cells were lysed at a density of 1.0×10⁸ cells/ml in lysisbuffer (1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 160 mM NaCl, 1 mMCaCl₂) containing complete protease inhibitor cocktail for 1 hour, andinsoluble material was pelleted at 10,000×g for 10 minutes. These andall subsequent steps were performed at 4° C. The lysate corresponding to5.0×10⁷ cells was incubated with 20 ul of protein G-Sepharose preloadedwith 10 ug of anti-PSGL-1 mAb (clone 2PH1, PharMingen, San Diego,Calif.), anti-TAIP mAb, TAB4, or IgG from normal hamster serum. Afterincubation for 4 hours at 4° C., the beads were washed five times withwashing buffer (0.05% Triton X-100, 50 mM Tris-HCl, pH 8.5, 400 mM NaCl,1 mM CaCl₂, 1 mg/ml ovalbumin), and twice with a similar washing buffer,containing 250 mM instead of 400 mM NaCl. Bound proteins were elutedwith 40 ul of 1×SDS sample buffer. Eluted proteins were separated by 6%SDS-PAGE and transferred to a nitrocellulose membrane. The membraneswere immunoblotted with anti-PSGL-1 mAb, and revealed byperoxidase-conjugated goat anti-rat IgG (H+L) followed bychemiluminescence (Renaissance, NEN).

[0136] Surface biotinylated RL♂1 T cells were lysed at a density of1.0×10⁸ cells/ml in lysis buffer. The cell extract was incubated with 20ug of antibody bound to 40 ul of protein G-Sepharose at 4° C. overnight.Depletions were done with anti-PSGL-1 mAb (2PH1) or control rat IgG,with TAB4 or control normal hamster serum. Depleted lysates were furthersubjected to do immunoprecipitation with TAB4 or anti-PSGL-1 mAb,respectively. Immunoprecipitates were separated on 6% SDS-polyacrylamidegel and detected by fluorography. As shown in FIG. 6, anti-PSGL-1antibody can deplete TAIP protein from T cell lysates. In addition,proteins immunoprecipitated with anti-TAIP antibody (TAB4) can berecognized by anti-PSGL-1 antibody by western analysis.

Example 10 Induction of Apoptosis in Human T Cells by an Anti-PSGL-1Antibody

[0137] To determine the role played by PSGL-1 in the apoptosis of humanT cells, time-course experiments were carried out to investigate whenactivated human T cells acquire sensitivity toward PSGL-1-mediatedapoptotic signals. Human T cells were stimulated with phytohemagglutinin(PHA) mitogen and further expanded in IL-2-containing medium. ActivatedT cells were harvested and then challenged with anti-PSGL-1 in thepresence of IL-2 and cross-linking antibodies.

[0138] Human peripheral blood was taken from healthy adults, heprinized,and enriched for peripheral blood mononuclear cells (PBMC) based ondifferential density using Ficoll-Paque Plus (Pharmacia Biotech). ThePBMC were activated with 1% PHA (Life Technologies, GibcoBRL) for 48hours and subsequently maintained in recombinant human IL-2 (5 ng/ml)through the assay period. To assess the apoptosis-inducing ability ananti-human PSGL-1 antibody, the activated cells were treated with: (1) 1ug/ml of the anti-PSGL-1 antibody clone KPL-1 (BD PharMingen) pluscross-linker rabbit anti-mouse Ig (0.5 ug/ml) (Jackson ImmunoResearchLaboratories); (2) isotype control purified mouse Ig plus cross-linkerrabbit anti-mouse Ig; or (3) cross-linker rabbit anti-mouse Ig alone.After six hours of treatment, the percentage of early apoptotic cellswas determined by FACS, staining with anti-Annexin V (BD PharMingen) andPI (Sigma).

[0139] As shown in FIG. 8, signaling triggered by PSGL-1 using ananti-PSGL-1 antibody plus the crosslinker triggered significant level ofapoptosis in PHA-activated human PBMC (mainly T cells). The percentageof apoptotic cells increased from 8.5% on days 3 to 24% on day 8 inanti-PSGL1 treated cultures. Neither isotopic-matched control, nor thecross-linking antibodies alone, had any effect on these cells.

EXAMPLE 11 Use of Anti-PSGL-1 Agonist Antibody to Treat AutoimmuneDisease

[0140] Non-obese diabetic (NOD) mice, a well-accepted autoimmunediabetes animal, were bred under standard conditions. Spontaneousdiabetes developed in the NOD mice at the age of about 20 weeks. In theexperimental group, the mice received three doses of anti-PSGL-1antibody (TAB4) intraperitoneally at 300 μg per mouse at age of 14, 15and 17 weeks. Two additional injections with the same dose were given atthe ages of 24 and 26 weeks. The control group was given hamster Ig atthe same dose. Mice were monitored for glucose uria by Medi-Test Glucosestrips (Macherey-Nagel, Germany) twice every week after the age of 15weeks. Non-fasting urine glucose levels over 300 mg/dl for twoconsecutive measurements were considered diabetic.

[0141] As shown in FIG. 9, TAB4 (anti-PSGL-1) antibody treatment yieldedsignificant protection as compared with control antibody treatment. Thusan anti-PSGL-1 antibody treatment can dampen the activity of autoimmuneT cells and delay the onset of type I-diabetes.

Example 12 Binding of P-Selectin, E-Selectin, and L-Selectin toActivated T Cells

[0142] To determine ability of selectins (P-Selectin, E-Selectin, andL-Selectin) to bind to activated T cells, freshly prepared splenocytesfrom C57BL/6 mice were activated and harvested at days 2, 4, and 6.Non-activated T cells (i.e., freshly prepared splenocytes at day 0) werealso analyzed. The day 2 sample constituted 3×10⁶ cells/ml ofsplenocytes that were activated with 2 ug/ml of Concanavalin A (Con A)in DMEM+10% FBS for 2 days. Live cells were isolated by Ficoll gradientseparation. The day 4 sample was obtained from cells that were activatedwith Con A for 3 days and maintained in medium containing 5 ng/ml ofIL-2 for an additional day. The day 6 sample was derived from cells thatwere activated with Con A for 3 days and maintained in 5 ng/ml of IL-2for 3 days.

[0143] To assay the samples by FACS analysis, 2×10⁵ cells per well fromdays 0, 2, 4 and 6 were incubated at 4° C. for 30 minutes with 40ul/well of mouse P-Selectin, E-Selectin, or L-Selectin fused to the Fcregion of human IgG1 (R&D Systems, Minneapolis, Minn.) at concentrationsranging from 20 ug/ml with two-fold serial dilution to 0.156 ug/ml.Following the incubation, cells were washed with 1× FACScan buffer(1×PBS without calcium and magnesium ions from Biochrom AG, Berlin and2% FBS). Samples were further incubated at 4° C. for 30 minutes with 95ul/well of anti-Thy1.2 and a secondary reagent (FITC-anti human IgG,which is specific to Fc fragment, purchased from Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.) at 3.25 ug/ml, and then washed with1× FACScan buffer.

[0144] The results of FACSCalibur analysis are shown in FIG. 10. At 20ug/ml, binding of P-selectin to mouse activated T cells increasedgradually, peaked on day 4, and declined slightly on day 6. Binding ofE-selectin rose significantly from day 2 to day 4 and then remainedpeaked at day 6. Binding of L-selectin to mouse activated T cells wasnot apparent, and did not change through the activation period, i.e.from day 0 to day 6. The results observed with L-Selectin could be dueto the apparent low binding affinity of L-selectin to its ligand.Similar results were also obtained when lower concentrations of thethree selectins were employed in the experiments.

Example 13 Multimeric Forms of E-Selectin and P-Selectin InduceApoptosis of Activated T Cells

[0145] A 96-well plate (NUNC) was coated with 50 ul of anti-human Fc Igat 20 ug/ml in 1×PBS at 4° C. overnight, blocked with 1% BSA at 37° C.for 2 hours and incubated with 50 ul of a selectin-human Fc fusion (from0.063 to 5 ug/ml) at room temperature for 2 hours. In all experimentalsteps, each well was thoroughly washed five times with 1×PBS. Then 2×10⁵T cells activated previously with Con A for four days were added intoeach well and incubated at 37° C. for 5 hours prior to centrifugation ofthe plate at 200×g for 5 minutes at 4° C. The resulting pelletcontaining activated T cells was incubated with Annexin V-biotinconjugate at room temperature for 15 minutes and subsequently with anavidin conjugate (SA-beta-gal at 1:5000 dilution) for another 30 minutesat 37° C. In every binding reaction, each well was washed thrice withAnnexin V binding buffer. The color development was achieved byincubating both 110 ul of Z-buffer mixture (54 ul of 2-mercaptoethanolin 20 ml of Z-buffer) and 30 ul of ONPG (0.04 g/10 ml) at 4° C.overnight. The readings of optical density at 420 nm were recorded.

[0146] Levels of selectin-induced apoptosis of Con-A activated T cellsincreased with the increasing concentrations (from 0.063 ug/ml to 5ug/ml) of P-selectin (FIG. 11A) or E-selectin (FIG. 11B) fused with Fcof human IgG1. The hamster antibody TAB4 induces apoptosis of activatedT cells (see Example 1) and was used as a positive control in theseexperiments. As negative controls, anti-human Fc, human Ig (HIg), andBSA did not induce apoptosis. No significant apoptosis was detected inthe presence of the L-selectin human Fc fusion protein (FIG. 11C),consistent with the failure of L-selectin to bind well to activated Tcells (Example 12).

[0147] In summary, a plate-bound fusion protein containing aPSGL-1-binding fragment of P-selectin or E-selectin and human Fcfragment induced apoptosis of activated T cells.

EXAMPLE 14 Cross-Linking of Soluble P-Selectin-Fc Fusion Protein InducesApoptosis of Activated T Cells

[0148] Mouse selectins (P-Selectin, E-Selectin, and L-Selectin) werefused to the Fc region of human IgG1 as detailed above to form solubledimeric fusion proteins. To evaluate whether the soluble selectins caninduce apoptosis of activated T cells, an experiment was performed asdetailed in the in Example 13, with the exception that the plate-boundanti-human Fc Ig was omitted. Negligible or low levels of apoptosis ofactivated T cells occurred in the presence of the soluble form ofP-selectin fusion protein (a dimer) alone (FIG. 12). However, upon theaddition of a cross-linker (anti-human Fc) the apoptotic activityincreased substantially, to approximately the apoptotic level induced inthe presence of the plate-bound antibody. Neither anti-human Fc, humanIg (HIg), nor BSA induced apoptosis.

[0149] Similar results were obtained for the E-selectin-Fc fusionprotein as were obtained for the P-selectin-Fc fusion protein. Inaddition, consistent with the results obtained for the plate-bound(multimeric form) of L-selectin, the soluble form of L-selectin fusionprotein did not induce apoptosis of activated T cells.

Other Embodiments

[0150] It is to be understood that, while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention. Other aspects, advantages, and modifications of theinvention are within the scope of the claims set forth below.

1 2 1 6 PRT Artificial Sequence synthetic peptide 1 Ile Glu Gly Arg MetAsp 1 5 2 6 PRT Artificial Sequence synthetic peptide 2 His His His HisHis His 1 5

What is claimed is:
 1. A method of preventing or reducing a Tcell-mediated immune response in an individual, the method comprising:selecting an individual diagnosed as having or as being at risk ofacquiring a condition characterized by an excessive or unwanted Tcell-mediated immune response; and administering to the individual amultimeric compound that binds to at least two P-Selectin GlycoproteinLigand 1 (PSGL-1) proteins on the surface of a T cell, wherein themultimeric compound comprises two polypeptide chains, each of thepolypeptide chains comprising (i) a binding domain that binds to PSGL-1,and (ii) a heterologous amino acid sequence, wherein the polypeptidechains are linked via the heterologous amino acid sequence to form themultimeric compound, wherein the binding of the multimeric compound tothe at least two PSGL-1 proteins on the surface of the T cell induces asignal transduction pathway that results in the death of the T cell,thereby preventing or reducing a T cell-mediated immune response in theindividual.
 2. The method of claim 1, wherein the multimeric compound isa homo-multimeric compound.
 3. The method of claim 1, wherein themultimeric compound is a hetero-multimeric compound.
 4. The method ofclaim 1, wherein the heterologous amino acid sequence comprises a cellsurface receptor binding region.
 5. The method of claim 1, wherein thebinding domain comprises a P-Selectin extracellular domain or aPSGL-1-binding fragment thereof.
 6. The method of claim 1, wherein thebinding domain comprises an E-Selectin extracellular domain or aPSGL-1-binding fragment thereof.
 7. The method of claim 1, wherein thebinding domain comprises an L-Selectin extracellular domain or aPSGL-1-binding fragment thereof.
 8. The method of claim 1, wherein thebinding domain comprises an antigen binding domain of an anti-PSGL-1antibody or a fragment thereof.
 9. The method of claim 1, wherein thebinding domain comprises a PSGL-1 binding polypeptide selected from aphage display library.
 10. The method of claim 1, wherein thepolypeptide chains are covalently linked via the heterologous amino acidsequence to form the multimeric compound.
 11. The method of claim 10,wherein the covalent linkage is a disulfide linkage.
 12. The method ofclaim 1, wherein the heterologous amino acid sequence comprises animmunoglobulin heavy chain constant region.
 13. The method of claim 1,further comprising administering to the individual an agent that bindsto the multimeric compound via the heterologous amino acid sequence andinduces cross-linking of a plurality of PSGL-1 antigens on the surfaceof the T cell.
 14. The method of claim 1, comprising selecting anindividual diagnosed as having an inflammatory disease.
 15. The methodof claim 1, comprising selecting an individual diagnosed as having anautoimmune disease.
 16. The method of claim 1, comprising selecting anindividual that has received or is expected to receive an allogeneic orxenogeneic transplant.
 17. The method of claim 1, comprising selectingan individual diagnosed as having an allergic disease.
 18. The method ofclaim 1, comprising selecting an individual diagnosed as having a T cellcancer.
 19. The method of claim 1, wherein the T cell is an activated Tcell.
 20. The method of claim 1, wherein the method comprises detectingthe number of T cells in a first biological sample taken from theindividual before the administration of the multimeric compound andcomparing the results with the number of T cells in a second biologicalsample taken from the individual after the administration of themultimeric compound.
 21. The method of claim 1, wherein the methodcomprises detecting a biological activity of T cells in a firstbiological sample taken from the individual before the administration ofthe multimeric compound and comparing the results with the biologicalactivity of T cells in a second biological sample taken from theindividual after the administration of the multimeric compound.
 22. Themethod of claim 1, wherein the administration results in the depletionof at least 10% of activated T cells in the individual.
 23. A method ofinducing the death of a T cell or a natural killer (NK) cell, the methodcomprising: providing a T cell or NK cell expressing PSGL-1 on its cellsurface; and contacting the T cell or NK cell with a multimeric compoundthat binds to at least two PSGL-1 proteins on the surface of the T cellor NK cell, wherein the multimeric compound comprises two polypeptidechains, each of the polypeptide chains comprising (i) a binding domainthat binds to PSGL-1, and (ii) a heterologous amino acid sequence,wherein the polypeptide chains are linked via the heterologous aminoacid sequence to form the multimeric compound, wherein the binding ofthe multimeric compound to the at least two PSGL-1 proteins on thesurface of the T cell or NK cell induces a signal transduction pathwaythat results in the death of the T cell or NK cell.
 24. The method ofclaim 23, wherein the multimeric compound is a homo-multimeric compound.25. The method of claim 23, wherein the multimeric compound is ahetero-multimeric compound.
 26. The method of claim 23, wherein theheterologous amino acid sequence comprises a cell surface receptorbinding region.
 27. The method of claim 23, wherein the binding domaincomprises a P-Selectin extracellular domain or a PSGL-1-binding fragmentthereof.
 28. The method of claim 23, wherein the binding domaincomprises an E-Selectin extracellular domain or a PSGL-1-bindingfragment thereof.
 29. The method of claim 23, wherein the binding domaincomprises an L-Selectin extracellular domain or a PSGL-1-bindingfragment thereof.
 30. The method of claim 23, wherein the binding domaincomprises an antigen binding domain of an anti-PSGL-1 antibody or afragment thereof.
 31. The method of claim 23, wherein the binding domaincomprises a PSGL-1 binding polypeptide selected from a phage displaylibrary.
 32. The method of claim 23, wherein the polypeptide chains arecovalently linked via the heterologous amino acid sequence to form themultimeric compound.
 33. The method of claim 32, wherein the covalentlinkage is a disulfide linkage.
 34. The method of claim 23, wherein theheterologous amino acid sequence comprises an immunoglobulin heavy chainconstant region.
 35. The method of claim 23, further comprisingcontacting the multimeric compound an agent that binds to the multimericcompound via the heterologous amino acid sequence and inducescross-linking of a plurality of PSGL-1 antigens on the surface of the Tcell.
 36. The method of claim 23, comprising inducing the death of anactivated T cell.
 37. The method of claim 23, wherein the methodcomprises assessing the viability of the T cell or NK cell after thecontacting with the multimeric compound.
 38. The method of claim 23,wherein the method comprises assessing a biological activity of the Tcell or NK cell after the contacting with the multimeric compound.
 39. Akit comprising: a multimeric compound that binds to at least two PSGL-1proteins on the surface of a T cell, wherein the multimeric compoundcomprises two polypeptide chains, each of the polypeptide chainscomprising (i) a binding domain that binds to PSGL-1, and (ii) aheterologous amino acid sequence, wherein the polypeptide chains arelinked via the heterologous amino acid sequence to form the multimericcompound, wherein the binding of the multimeric compound to the at leasttwo PSGL-1 proteins on the surface of the T cell induces a signaltransduction pathway that results in the death of the T cell; andinstructions for use of the multimeric compound to treat inflammation,autoimmunity, transplant rejection, an allergic condition, or a T cellcancer.